(A) Cytokine levels in CE supernatants after 24 hours of culture (ELISA). Represented values are normalized to milligrams of cultured colonic tissue. (B and C) Cytokine levels obtained from LPLs (B) and IELs (C) isolated from the colons of Il10^–/– and Il10^–/–Tlr4^–/– mice and stimulated with anti-CD3/CD28 Abs for 24 hours Data represent mean ± SEM of 3 different experiments. *P < 0.05, **P < 0.01.

TLR4 triggering of CD4⁺ T cells activates NF-κB and MAPK pathways. However, the induction of MKP1 and MKP3 occurs mainly via the TRIF pathway. The high levels of MKP-1 and MKP-3 restrain a subsequent TCR-dependent activation of p38 and ERK, respectively. This activation of phosphatases in CD4⁺ T cells by TLR4 restrains subsequent TCR-induced phosphorylation events and, hence, modulates CD4⁺ T cell responses and their inflammatory phenotypes. AP-1, activator protein 1; MKK, MAPK kinase.

(A) Histological analysis of 8-week-old B6, Il10^–/–, Il10^–/–Tlr9^–/–, and Il10^–/–Tlr4^–/– mice maintained under specific pathogen–free conditions showing increased epithelial crypt hyperplasia in Il10^–/–Tlr4^–/– mice (original magnification, ×100). (B) Cross-sectional analysis of colons from the different mice showing greater cellular infiltration in the mice lacking TLR4 (original magnification, ×100). (C) Quantitative measurement of crypt length and cellular infiltration in the different groups of mice at 8 weeks of age. (D) Histological analysis of 4-week-old Il10^–/– mice housed with or without 7- to 8-week-old Il10^–/–Tlr4^–/– mice (original magnification, ×100). (E) Quantitative measurements of crypt length and cellular infiltration in co-housed animals. Data represent mean ± SEM of 3 different experiments. **P < 0.01.

(A) qPCR analysis of Tlr4 expression in unstimulated CD4⁺ T cells from spleens (Sp), MLNs, LPLs, and IELs of Il10^–/– mice. Total splenocytes (Total Sp) were used as a positive control. (B) qPCR analysis of Tlr4 expression in spleen CD4⁺ T cells before and 24 hours after anti-CD3/CD28 Abs stimulation. (C and D) MLN CD4⁺ T cells were treated with LPS for the indicated times. Nuclear lysates were examined for NF-κB activation (EMSA) (C), and cytosolic lysates were tested for ERK1/2, p38, and JNK activation by immunoblotting with phospho-specific Abs (D). (E and F) NF-κB and ERK activation in Tlr4^–/– MLN CD4⁺ T cells after LPS stimulation. All CD4⁺ T cells used were FACS sorted, and purity was greater than 98% in all experiments. Stimulation was carried out with 100 ng/ml of LPS in all experiments. Data represent mean ± SEM and are representative of 3 independent experiments (4 mice per experiment were used in A and B). *P < 0.05, **P < 0.01.

(A and B) Percentage of initial body weight of Rag1^–/– recipients transferred with Il10^–/– and Il10^–/–Tlr4^–/– FACS-sorted naive CD45RB^hi T cells (A) and cotransferred with regulatory CD45RB^loCD25⁺ Tregs (B6) (B). (C) Microscopic evaluation revealed mild inflammation in the colons of Il10^–/– CD45RB^hi recipients, while severe inflammation was observed in the colon of Il10^–/–Tlr4^–/– CD45RB^hi recipients (original magnification, ×100). (D and E) Quantitative analyses of crypt length and cellular infiltration performed in colonic tissues from the recipient mice. Asterisks represent significant differences compared with control group (group receiving Il10^–/– naive and B6 Tregs). (F) Cytokine levels in CE supernatants after 24 hours of culture. (G) CD4⁺ T cell cytokine response. CD4⁺ cells from colon (IELs and LPLs), MLNs, and spleen were isolated from Rag1^–/– recipients and stimulated with anti-CD3/CD28 Abs. Cytokine levels were determined 24 hours later. Data represent mean ± SEM of 2 independent experiments. *P < 0.05, **P < 0.01.

(A) Splenic CD4⁺ T cells from OT-II mice were incubated for 2 hours in the presence of 100 ng/ml LPS or medium alone. The cells were then washed and cultured for 5 days with OVA-loaded BMDCs. After coculture, the CD4⁺ cells were restimulated, and the secretion of cytokines was determined 24 hours later. (B) NF-κB activation was assessed in nuclear extracts by EMSA. (C) Cytosolic lysates were immunoblotted for MAPK protein activation after anti-CD3/CD28 Ab stimulation with or without LPS (100 ng/ml) for 2 hours prior to TCR stimulation. (D) LPS-dependent regulation of ERK1/2 phosphorylation was confirmed by phospho-protein analysis by flow cytometry as described in Methods. Numbers within histograms denote the percentage of p-ERK1/2–positive cells in stimulated cells when compared with control cells. (E) Activation of NFAT-1 was measured by immunoblotting of nuclear extracts from CD4⁺ cells stimulated as in C. (F) RT-PCR analysis of CD4⁺ T cells incubated with the MEK/ERK1/2 inhibitor UO126 or vehicle (DMSO) before being stimulated with anti-CD3/CD28 Abs for the indicated times. Data in A–F represent mean ± SEM of 3 independent experiments. *P < 0.05. All experiments, except in A, were carried out with FACS-sorted MLN-derived CD4⁺ cells from Il10^–/– mice (purity, >98%).

(A) Immunoblot analysis of the indicated phosphatases after LPS stimulation of CD4⁺ cells at different time points. (B) Expression of MKPs and p-SHIP1 in freshly isolated, unstimulated CD4⁺ cells from Il10^–/– and Il10^–/–Tlr4^–/– mice. (C) Immunoblotting of protein extracts from CD4⁺ T cells stimulated with LPS or left untreated before stimulation with anti-CD3/28 Abs for different periods of time. (D) Analysis of the siRNA knockdown in CD4⁺ T cells 24 hours after transfection with either MKP-3 or control siRNA (Ctrl). (E) TCR-dependent phosphorylation of ERK1/2 24 hours after transfection with MKP-3 or control siRNA. Numbers within histograms denote percentage of p-ERK1/2–positive cells in stimulated cells when compared with control cells. (F) Cytokine levels measured from 24-hour supernatants of cells treated as in E. Data represent mean ± SEM. (G) p-ERK expression in freshly isolated CD4⁺ cells from Il10^–/– mice treated with MKP inhibitor in vivo (NSC 95397) or vehicle. (H) Cytokine levels after 24 hours of anti-CD3/28 Ab stimulation of MLN-derived CD4⁺ T cells isolated from Il10^–/– mice 3 days after injection of NSC 95397 or vehicle. Data represent mean ± SEM. Data are representative of at least 2 independent experiments. *P < 0.05. All experiments were carried out with FACS-sorted MLN-derived CD4⁺ cells from Il10^–/– or Il10^–/–Tlr4^–/– mice (purity, >98%). Stimulation was carried out with 100 ng/ml LPS in all experiments. The lanes shown in B were run on the same gel but were noncontiguous.